national centre for biotechnology education nature’s dice copyright © dean madden, 2010
Post on 20-Dec-2015
213 views
TRANSCRIPT
National Centre for Biotechnology Education
www.ncbe.reading.ac.uk
Nature’s dice
Copyright © Dean Madden, 2010
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Sickle cell
Normal red blood cells
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Dominant allele (D)
Recessive allele (d)
DNA remains uncut
DNA is cut
Restrictionenzyme
6500 base-pairs (bp)
2500 bp 4000 bp
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Summary of procedure
DNA
Enzyme
Loading dye
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Microsyringe
Graduated tip
HOLD HEREDo not touch the point!
10 µL
2 µL
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Mass A microgram is one millionth of a gram
1 000 micrograms (µg) = 1 milligram (mg) 1 000 milligrams = 1 gram (g)
Volume
A microlitre is one millionth of a litre 1 000 microlitres (µL) = 1 millilitre (mL)
1 000 millilitres = 1 litre (L)
Cop
yright ©
Dea
n M
ad
de
n, 20
10
IMPORTANTMix well after dispensing
20 µL
Numbered DNA sample
Dried restriction enzyme in tube
Label the tube twice (on the side and on
the lid)
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Close the tubes and push them firmly into the foam floater
Incubate for 30–45 minutes at 37°C
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Frosted panel on this side
Molten agarose55–60 °C
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Cut two electrodes
Carbon fibretissue
42 mm
22 mm
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Pour 2–3 mm depth of buffer over the gel before you ease the comb out
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Mix loading dye into each
DNA sample
2 µL
Bromophenol blue loading dye
Cut DNA sample
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Label the end of the tank to show the contents of
each well
Black card under the tank reveals
the wells for loading
Load the DNA through the buffer, taking care not to puncture the wells as you do so
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Electrodes
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Direction of DNA movement
Place a comb over the tank to reduce
evaporation
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Leave the stain on for 4 minutes only
Azure A stain
Cop
yright ©
Dea
n M
ad
de
n, 20
10
DNA
Positively-charged Azure A binds to the negatively-charged
phosphate groups of the DNA
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Area with DNA bands
WellsLoading
dye
Compare each band from each DNA sample with bands of known sizes in the DNA ‘ruler’
Cop
yright ©
Dea
n M
ad
de
n, 20
10
DNA ‘ruler’ or ‘ladder’
Cop
yright ©
Dea
n M
ad
de
n, 20
10
Unaffected Affected Carrier